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Plasmodium falciparum Histidine-rich Protein-2 (PfHRP2) Modulates the Redox Activity of Ferri-protoporphyrin IX (FePPIX) : PEROXIDASE-LIKE ACTIVITY OF THE PfHRP2-FePPIX COMPLEX Ryuichi Mashima, Leann Tilley, Mary-Anne Siomos, Vicki Papalexis, Mark J. Raftery and Roland Stocker
J. Biol. Chem. 2002, 277:14514-14520. 10.1074/jbc.M109386200 originally published online February 21, 2002 doi:

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THE JOURNAL OF BIOLOGICAL CHEMISTRY 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 17, Issue of April 26, pp. 1451414520, 2002

Printed in U.S.A.

Plasmodium falciparum Histidine-rich Protein-2 ( PfHRP2) Modulates the Redox Activity of Ferri-protoporphyrin IX (FePPIX)
PEROXIDASE-LIKE ACTIVITY OF THE PfHRP2-FePPIX COMPLEX*
Received for publication, September 28, 2001, and in revised form, February 18, 2002 Published, JBC Papers in Press, February 21, 2002, DOI 10.1074/jbc.M109386200

Ryuichi Mashima, Leann Tilley, Mary-Anne Siomos, Vicki Papalexis, Mark J. Rafteryll, and Roland Stocker**
From the Biochemistry Group, The Heart Research Institute, 145 Missenden Road, Camperdown, New South Wales 2050, the Department of Biochemistry, La Trobe University, Bundoora, Victoria 3086, and the IlCytokine Research Unit, School of Pathology, University of New South Wales, Kensington, New South Wales 2052, Australia

Histidine-rich protein-2 from Plasmodium falciparum (PfHRP2) binds up to 50 molecules of ferri-protoporphyrin IX (FePPIX) (Choi, C. Y., Cerda, J. F., Chu, H. A., Babcock, G. T., and Marletta, M. A. (1999) Biochemistry 38, 1691616924). We reasoned that the PfHRP2-FePPIX complex has antioxidant properties that could be beneficial to the parasite. Therefore, we examined whether binding to PfHRP2 modulated the redox properties of FePPIX. We observed that PfHRP2 completely inhibited the auto-oxidation of ascorbate mediated by free FePPIX. We also investigated the peroxidase activity of PfHRP2FePPIX using 13-hydroperoxy-9,11-octadienoate (18:2-OOH) as substrate. Reaction of PfHRP2-FePPIX with 18:2-OOH in the presence of added reducing agents gave 13-hydroxy-9,11octadienoate (18:2-OH) as a major product and 13-keto-9,11octadienoate (18:2=O) and 9,12,13-trihydroxy-10octadecaenoate as minor products. Binding of FePPIX to PfHRP2 lowered the rate of decomposition of 18:2-OOH and increased the 18:2-OH to 18:2=O ratio. Similar to other authentic peroxidases, phenols, amines, and biological reductants like ascorbate promoted 18:2-OH production, and NaCN inhibited 18:2-OH production. Thioanisole also acted as a reductant and was converted to thioanisole sulfoxide, suggesting formation of compound I during the reaction. These data show that PfHRP2 modulates the redox activity of FePPIX and that the PfHRP2-FePPIX complex may have previously unrecognized antioxidant properties.

site (2), corresponding to -15 mmol/liter H2O2 being produced. H2O2 is a potentially harmful oxidant that can cause damage to protein and lipid in the presence of transition metals and reducing substances. Indeed, parasitized red cells are exposed to oxidative stress (reviewed in Ref. 3).

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During the blood stage of malaria infection, the parasite feeds on red cell proteins, principally hemoglobin. Degradation of hemoglobin takes place in the food vacuole, and this process is initiated by the conversion of oxyhemoglobin to methemoglobin with the concomitant production of superoxide anion that dismutates to H2O2 (1). About 80% of the hemoglobin is degraded during the intra-erythrocytic development of the para* This work was supported by the Australian National Health and Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Present address: Centre for Thrombosis and Vascular Research, School of Medical Sciences, University of New South Wales, UNSW Sydney, New South Wales 2052, Australia. ** To whom correspondence should be addressed: Centre for Thrombosis and Vascular Research, School of Medical Sciences, University of New South Wales, UNSW Sydney, New South Wales 2052, Australia. E-mail: r.stocker@unsw.edu.au.

A second toxic insult comes from ferri-protoporphyrin IX (FePPIX)1 released as the byproduct of methemoglobin degradation (1). Free FePPIX is toxic because of its detergent-like (4) and redox properties (5). The parasite detoxifies FePPIX via nonenzymatic processes, including reaction with H2O2 and glutathione, that lead to the accumulation of iron (6). In addition, FePPIX is crystallized to a granular pigment, known as hemozoin (f3-hematin) (7). This process is initiated and accelerated by histidine-rich proteins, of which Plasmodium falciparum histidine-rich protein-2 ( PfHRP2) is characterized best. Histidine comprises 34% of the amino acid residues of PfHRP2 (8, 9), and for effective conversion of FePPIX to f3hematin, the protein requires an acidic pH that exists in the food vacuole (10, 11). PfHRP2 is also present outside the food vacuole, i.e. underneath the red cell membrane and in the erythrocyte cytoplasm (12). This indicates a biological role for PfHRP2 at neutral pH where it acts as an efficient FePPIX-binding protein rather than promoting f3-hematin formation (11). Up to -50 mol of FePPIX binds tightly per mol of PfHRP2 through hexa-coordination (13) to the repetitive amino acid sequence AHHAHHAAD (14). Although the spectroscopic properties of the PfHRP2-FePPIX complex are well characterized (13), its biochemical properties are poorly understood. We reasoned that the coordinated binding to PfHRP2 modulates the redox activity of FePPIX and that the PfHRP2-FePPIX complex may possess peroxidase activity. FePPIX-containing peroxidases reduce hydroperoxides (ROOH) via heterolysis to the corresponding alcohol (ROH) and compound I (Reaction 1), an intermediate with an oxidation state two electrons higher than that of the resting enzyme. In contrast, free FePPIX and the heme proteins cytochrome P-450 and hemoglobin decompose ROOH through homolysis to
1 The abbreviations used are: FePPIX, ferri-protoporphyrin IX; 18: 2=O, 13-ketooctadeca-9,11-dienoic acid; 18:2-OH, 13-hydroxyoctadeca9,11-dienoic acid; 18:2-OOH, 13-hydroperoxyoctadeca-9,11-dienoic acid; 20:4-OOH, 15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid; HPLC, high-performance liquid chromatography; OPD, o-phenylenediamine; PBS, phosphate-buffered saline; PfHRP2, P. falciparum histidine-rich protein-2; PLPC, 1-palmitoyl-2-linoleoyl phosphatidylcholine; PLPCOOH, 1-palmitoyl-2-linoleoyl phosphatidylcholine hydroperoxide; PPIX, protoporphyrin IX; SnPPIX, tin PPIX; ZnPPIX, zinc (II) PPIX; ROOH, hydroperoxide; ROH, corresponding alcohol; RO ~, alkoxyl radical; AH 2,

hydrogen donor.

Plasmodium falciparum Histidine-rich Protein-2

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FIG. 1. Auto-oxidation of ascorbate. Ascorbate (100 AM) was incubated at 37 C in PBS containing 5 tM FePPIX (0), 1 AM PfHRP2 (), PfHRP2FePPIX (1 WM PfHRP2 and 5 AM FePPIX, ), and PBS (). The data are expressed as the means S.D. (n = 3).

alkoxyl radical (RO') and an oxo-ferryl species (Reaction 2) (15). Catalytic reduction of ROOH requires consecutive oneelectron reduction of the FePPIX peroxidase intermediates compound I (Reaction 3) and compound II (the one-electron oxidized form of the enzyme) (Reaction 4), and this is achieved by hydrogen donors (AH2) such as amines and phenols. Alternatively, compound I can be reduced by sulfur- and nitrogen-containing compounds such as thioanisole through oxygen transfer (Reaction 5) (16, 17).
ROOH+Fe(III)peroxidase>ROH+ compound I ROOH+Fe(III)>RO' +Fe(IV)= O Compound I+AH2>Compound II+AH' Compound II+AH2>Fe(III)peroxidase+AH' Compound I +X>Fe(III)peroxidase +X=O REACTIONS 15

In the present study, we investigated the redox properties of the PfHRP2-FePPIX complex. We show that binding to PfHRP2 decreases the pro-oxidant properties and increases the peroxidase activity of FePPIX, raising the possibility that PfHRP2 acts as a previously unrecognized antioxidant by atten uating alkoxyl and hydroxyl radical formation.
EXPERIMENTAL PROCEDURES

Materials Linoleic acid, arachidonic acid, o-phenylenediamine (OPD), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), GSH, N, N'-diphenyl-1,4-phenylenediamine, phenol, gallic acid, salicylic acid,

1,2,4-aminotriazole, ferri-protoporphyrin IX (hematin, FePPIX), and thioanisole were obtained from Sigma. NaCN and NaN 3 were from Merck, 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) was from Avanti Polar-Lipids Inc. (Alabaster, AL), and guaiacol was from Fluka (Buchs, Switzerland). Bilirubin conjugate, protoporphyrin IX (PPIX), and tin protoporphyrin IX (SnPPIX) were from Porphyrin Products (Logan, UT), and Trolox and zinc (II) protoporphyrin IX (ZnPPIX) from Aldrich. [1-14C]Linoleic acid (55 mCi/mmol) and PD-10 columns were from Amersham Biosciences. Unless specified otherwise, aqueous buffers were stored over Chelex-100 (Bio-Rad) prior to use. 13-Hydroxy9,11octadienoate (18:2-OH) was prepared from 18:2-OOH as described (18). 13-Keto-9,11-octadienoate (18:2 =O) was prepared by reduction of 18:2OOH with acetylchloride in pyridine or purchased from Cayman (Ann Arbor, MI). Expression and Purification of Recombinant PfHRP2 BL21/DE3 Escherichia coli (Stratagene, La Jolla, CA) transfected with the pET-8c expression vector containing the recombinant Pf HRP2 sequence (kindly donated by Drs. D. Sullivan and D. Goldberg, Howard Hughes Medical Institute) were grown overnight, and protein expression was induced by isopropyl- 3-D-thiogalactopyranoside (0.84 m M). After incubation at 37 C for 4 h, the harvested cells were sonicated (XL2000, Daintree), and PfHRP2 was purified from the supernatant using a Ni 2+-agarose column (Novagen) eluted with 20 m M Tris-HCl (pH 7.9) containing 1 M imidazole. The recombinant PfHRP2 used in this study contains 93 histidine residues and binds 46 molecules of FePPIX/protein (13). Un-

FIG. 2. Degradation of 18:2-OOH by FePPIX and PfHRP2-FePPIX. 18:2-OOH (5 AM) was reacted at 37 C with FePPIX alone () or PfHRP2-FePPIX (1 WM PfHRP2 and 1 tM FePPIX, ) in 50 mM sodium phosphate buffer (pH 7.2) in the presence of 100 toM OPD. At the times indicated, the 18:2-OOH concentration was determined as described under Experimental Procedures. The data are expressed as the means S.D. (n = 3). Where error bars cannot be seen, they are smaller than the size of the symbols. Downloaded from http://www.jbc.org/ by guest on June 23, 2013

less otherwise specified, reconstitution was performed for 5 min at 37 C in 50 m M sodium phosphate buffer (pH 7.2) containing equimolar amounts of Pf HRP2 and FePPIX (13). For some experiments Pf HRP2 (1.1 A M ) was reconstituted with different metallo-porphyrins (100 A M ) in 200 m M sodium phosphate buffer (pH 7.2) for 5 min at 37 C. Excess metallo-porphyrin was then removed by gel filtration (PD-10 column) using PBS as eluent. Preparation of Lipid Hydroperoxides15-Hydroperoxyeicosa5,8,11,13tetraenoic acid (20:4-OOH), 18:2-OOH, and 1-palmitoyl-2-linoleoyl phosphatidylcholine hydroperoxide (PLPC-OOH) were prepared by lipoxygenase-catalyzed oxidation and purified by solid phase extraction (C18 cartridge, Waters, Milford, MA) followed by preparative reversedphase HPLC using an LC-18 column (20 X 250 mm, 5 Am, Supelco, Bellofonte, PA) (19). 18:2-OOH and 20:4-OOH were eluted with methanol/water/acetic acid (950:50:1), whereas PLPC-OOH was eluted using 0.02% triethylamine in methanol at 8.0 ml/min. [1-14C]18: 2-OOH (55 mCi/mmol) was prepared similarly, except that an analytical LC-18 column (4.6 X 250 mm, 5 Am, Supelco) was used, and the flow rate of the mobile phase was 1.0 ml/min. The fractions containing the respective hydroperoxide were collected, dried, and redissolved in methanol for determination of the concentration at 234 nm using = 28,000 cm' (20).

Auto-oxidation and Co-oxidation Experiments The time-dependent decay of ascorbate was used to examine the ability of PfHRP2 to render catalytic metals inactive (21). Briefly, ascorbate (100 AM) was incubated at 37 C in nonchelexed PBS containing the additives specified, and the decay of ascorbate followed at 265 nm. For co-oxidation experiments, PLPC (100 oM) was incubated in PBS containing 1 AM PfHRP2, 5 AM FePPIX, 100 AM OPD at 37 C, and the time-dependent formation of PLPC-OOH was determined as described below. Peroxidase Activity ofPfHRP2The time-dependent accumulation of 18:2-OH and 18:2=O from 5 18:2-OOH was monitored at 37 C in PBS containing 1 mM OPD and PfHRP2 reconstituted with FePPIX or the metallo-porphyrin indicated. Control experiments contained either FePPIXfree PfHRP2, FePPIX alone, or water as vehicle. At the times indicated, the concentrations of 18:2-OH, 18:2-OOH, and 18:2 =O were determined as described below. Where indicated, NaCN, NaN 3, or 1,2,4- aminotriazole (0 10 mM) was added, in which case the peroxidase activity of 1 AM PfHRP2 and 1 FePPIX was examined using 100 AM 18:2-OOH and 500 AM OPD in 50 mM sodium phosphate (pH 7.2) and an incubation period of 15 min. Steady-state KineticsThe substrate specificity of FePPIX-bound PfHRP2 was examined for 5 min at 37 C in 50 m M phosphate buffer (pH 7.2) containing 1 AM PfHRP2, 1 AM FePPIX, 100 AM 18:2-OOH, and 0200 AM of the reducing substrate indicated. Km and Vmax were determined from the respective Michaelis-Menten plots obtained under the same conditions using 100 AM OPD and 0200 AM 18:2-OOH. Involvement of Compound I in Peroxidase Activity of PfHRP2 The involvement of compound I in the peroxidase activity of PfHRP2 was assessed by the conversion of thioanisole to thioanisole sulfoxide (15). Briefly, FePPIX-containing PfHRP2 (3 AM each) was incubated at 37 C in PBS containing 200 AM 18:2-OOH and 1.6 m M thioanisole. The concentration of thioanisole sulfoxide was determined by HPLC using an LC-18 column (4.6 X 150 mm, 3 Am, Supelco) eluted with methanol/ water (40:60) at 0.8 ml/min and monitored at 254 nm. Under these

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Plasmodium falciparum Histidine-rich Protein-2

T ABLE I Reaction of 18:2-OOH with PfHRP2-FePPIX The peroxidase-like activities of PfHRP2-FePPIX (0.1 AM PfHRP2 and 5 AM FePPIX), FePPIX (5 tM), or GSH peroxidase (0.16 AM in selenium) was determined following incubation for 5 min at 37 C in 50 mM sodium phosphate buffer (pH 7.2) using 200 AM 18:2-OOH and 1 mM reducing substrate (OPD for both PfHRP2-FePPIX and FePPIX and GSH for GSH peroxidase). Concentrations of 18:2-OOH and its metabolites were determined by HPLC on an LC18 column (4.6 X 150 mm, 3 Am) with acetonitrile/water/acetic acid (60:40:0.1) at 1.0 ml/min. Quantitation was performed by UV detection at 234 nm for 18:2-OH and 18:2-OOH and 280 nm for 18:2=O. The data are expressed as the means S.D. of three experiments.
Catalyst Products 18:2-OH
ttM

18:2=O
gM

18:2-OHb
(%)

18:2=Ob
(%)

18:2-OH

Turnover numbera 18:2=O

18:2-OH/18:2=O

PfHRP2-FePPIX 73 6 22 1 41 3 12 1 15 1 FePPIX 42 2c 27 6 22 1c 14 3 80 GSH peroxidase 94 11 NDd 86 11 NAe 2344 277 Turnover number was calculated by the amount of product per amount of catalyst. Yield of products (in percentages) was calculated by products formed per 18:2-OOH degraded. p < 0.05, PfHRP2-FePPIX versus FePPIX. ND, not detected. NA, not applicable.
a b d e

40 51 NA

3.4 0.4 1.6 0.2c NA

Downloaded from http://www.jbc.org/ by guest on June 23, 2013 Downloaded F IG . 3. Reversed-phase HPLC chromatograms of 18:2-OOH and its metabolites. A and B , 200 A M 18:2-OOH was added to 50 m M sodium phosphate buffer (pH 7.2) containing PfHRP2-FePPIX (0.1 A M PfHRP2 and 5 A M FePPIX) at 37 C for 5 min. Elution was performed isocratically with acetonitrile/water/acetic acid (60:40:0.1) at a flow rate of 1.0 ml/min on an LC-18 column (4.6 X 150 mm, 3 A m, Supelco). 18:2-OH and 18:2-OOH ( A) and 18:2 = O ( B ) were detected at 234 and 280 nm, respectively. C , 50 ti M [1- 14C]18:2-OOH (55 mCi/mmol) was reacted in PBS containing Pf HRP2-FePPIX (1 A M PfHRP2 and 1 A M FePPIX) at 37 C for 15 min. Radioactivity was measured using a radiometric detector after separation by reversedphase HPLC as described above. The ratio of flow rate of the mobile phase and scintillant was 3. Peaks 14 correspond to 18:2-OOH, 18:2OH, 18:2 = O, and 9,12,13-tirhydroxy-10-octadecaenoic acid, respectively (Scheme 1). D , mass spectrum of 9,12,13-tirhydroxy-10octadecaenoic acid obtained by collision-induced dissociation experiment using negative electrospray ionization. Mass spectrum was obtained by TSQ-7000 with 0.005% ammonium acetate in methanol/water (50:50) using m / z = 329.2 as a precursor ion. conditions, thioanisole sulfoxide (containing both diasteromers) and thioanisole eluted at 4.3 and 44 min, respectively. Quantification of thioanisole sulfoxide was by area comparison using an authentic standard prepared from thioanisole by oxidation with stoichiometric amounts of hydrogen peroxide. Analyses of Oxidized Lipids The concentrations of 20:4-OOH, 18:2 OOH, 18:2-OH, and 18:2=O were determined by HPLC on an analytical LC-18 column eluted with acetonitrile/water/acetic acid (60:40:0.1) at

FIG. 4. Co-oxidation of PLPC. Co-oxidation of 132 AM PLPC was carried out at 37 C in 50 m M sodium phosphate buffer (pH 7.2) containing 200 AM 18:2-OOH, 100 AM OPD, and either water (control, ), PfHRP2 (1 toM, ), FePPIX (1 toM, 0), or PfHRP2-FePPIX (1 WM PfHRP2 and 1 tM FePPIX, ). 1.0 ml/min. 20:4-OOH, 20:4-OH, 18:2-OOH, and 18:2-OH were detected at 234 nm, and 18:2 =O was detected at 280 nm, and the compounds were quantified using = 28,000 and 22,000 cm -1 for the conjugated diene and oxodiene, respectively. [1- 14C]18:2-OOH and its decomposition products were separated under the same chromatographic conditions, using a radiometric detector A140 (Canberra Packard, Meriden, CT) and UltimaGold (Canberra Packard) as scintillant. PLPC-OOH and its corresponding hydroxide were quantified at 234 nm after separation on an ODS column (4.6 X 250 mm, 5 Am; Phenomenex, Pennant Hills, Australia) eluted with acetonitrile/methanol/water (50:49.5:0.5) containing 10 m M choline chloride at 1.0 ml/min. Mass SpectrometryThe reaction products were analyzed by a TSQ7000 mass spectrometer (Finnigan MAT, San Jose, CA) operated in negative electrospray ionization mode with the capillary voltage set at -4.5 kV, capillary temperature at 180 C, and scan time of 2 s. The samples (10 p.l)

were injected by direct flow injection. The analytes were ionized using 0.005% ammonium acetate in methanol/water (50:50) at 10 tl/min. The following values of (deprotonated) masses were obtained: 18:2-OH, 295.0 (calculated, 295.5); 18:2=O, 293.0 (calculated, 293.4); 9,12,13trihydroxyocta-10-decaenoic acid, 329.2 (calculated, 329.5). For collisioninduced dissociation experiments, the collision energy was set at 56 eV, and 0.005% ammonium acetate was delivered at 5 Al/min. RESULTS

Previous studies showed that FePPIX bound to PfHRP2 is hexa-coordinated and bis-histidyl ligated (13). To test whether such binding modulates the redox properties of FePPIX, we first examined the effect of FePPIX on the auto-oxidation of ascorbate in the absence and presence of PfHRP2 in nonchelexed PBS. As expected, ascorbate auto-oxidized readily in the absence of PfHRP2 (Fig. 1). This auto-oxidation was inhibited by chelex treatment of the buffer (not shown), as reported previously (21), indicating that it was caused by catalytic metals present in PBS. The addition of PfHRP2 did not signifi-

potentially damaging alkoxyl radicals. Increasing the FePPIX to PfHRP2 ratio resulted in an increased extent of 18:2-OOH degradation (Fig. 5A) with unal-

Plasmodium falciparum Histidine-rich Protein-2 14517


Downloaded from http://www.jbc.org/ by guest on June 23, 2013 SCHEME 1. Mechanism of heterolysis and homolysis of 18:2- OOH (adapted from Ref. 22).

cantly alter the rate of ascorbate auto-oxidation (Fig. 1), indicating that the protein was not able to prevent metal-catalyzed oxidation of ascorbate. Inclusion of free FePPIX but not PfHRP2-ligated FePPIX enhanced the rate of ascorbate oxidation (Fig. 1), indicating that the protein can regulate the redox activity of FePPIX. FePPIX is the prosthetic group of several hydroperoxidemetabolizing enzymes. To examine further the redox property of PfHRP2-ligated FePPIX, we tested the peroxidase activity of the complex using 18:2-OOH as substrate and OPD as reducing agent. FePPIX rapidly decomposes 18:2-OOH to the corresponding hydroxy-, keto-, and trihydroxy-fatty acids (22), a finding we confirmed (Fig. 2). Decomposition was almost instantaneous (Fig. 2), with the hydroxy- and keto-fatty acids accounting for 22 and 14%, respectively, of the starting material (Table I). FePPIX and certain heme proteins cause both heteroand homolytic cleavage of 18:2-OOH to yield 18:2-OH and 18:2=O, respectively (22), whereas authentic peroxidases convert 18:2-OOH exclusively into 18:2-OH via heterolysis (Scheme 1). Thus, the ratio of 18:2-OH to 18:2 =O reflects the relative contribution of hetero- to homolytic degradation of 18:2OOH (22). Compared with FePPIX alone, the PfHRP2- FePPIX complex significantly lowered the rate of 18:2-OOH decomposition (Fig. 2) and increased both the yield of 18:2-OH and the ratio of 18:2-OH to 18:2 =O (Table I). This product distribution indicated an enhanced peroxidase-like activity of the PfHRP2-FePPIX complex compared with FePPIX alone, although it remained lower that that seen with GSH peroxidase (Table I). Similarly, the presence of PfHRP2 doubled the turnover number for 18:2-OH compared with FePPIX alone, although again the value for the PfHRP2-FePPIX complex remained lower than that of GSH peroxidase (Table I). The metal chelators, diethylenetriaminepentaacetic acid and EDTA, did not affect the peroxidase-like activity of the PfHRP2-FePPIX complex (not shown). A significant proportion of the 18:2-OOH degraded by the PfHRP2-FePPIX complex was not recovered as 18:2-OH or 18:2=O. Separate experiments employing [114 C]18:2-OOH as substrate revealed 9,12,13-trihydroxyoctadecaenoic acid as a additional product (Fig. 3). The above results further support the idea that PfHRP2 can modulate the redox activity of FePPIX and that it decreases the proportion of homolytic degradation of 18:2-OOH. The latter pathway is associated with the formation of alkoxyl radicals (Scheme 1) that can co-oxidize suitable substrates (23, 24). We therefore incubated PLPC with 18:2-OOH and FePPIX in the absence and presence of PfHRP2. As expected, the presence of FePPIX caused the time-dependent accumulation of PLPCOOH (Fig. 4). PfHRP2 inhibited this process by up to 50% (Fig. 4), demonstrating that the protein inhibited the formation of

F IG . 5. FePPIX-dependent formation of 18:2-OH and 18:2 = O from 18:2-OOH. A , FePPIX (05 M ) was reconstituted with 0.1 M Pf HRP2 and the formation of 18:2-OH ( ) and 18:2 = O ( + ) measured in 50 m M phosphate buffer (pH 7.2) containing 200 M 18:2-OOH and 1 m M OPD at 37 C for 5 min. B , ratio of 18:2-OH to 18:2 = O produced under the condition of varying FePPIX to Pf HRP2 ratio used in A . The concentrations of products were measured by HPLC as described under Experimental Procedures. The data are expressed as the means S.D. ( n = 3).

F IG . 6. Inhibition of the peroxidase activity of Pf HRP2-FePPIX. M Pf HRP2 and 1 M FePPIX) was pretreated with increasing concentrations of NaCN ( ), NaN 3 ( 0 ), or 1,2,4-aminotriazole ( E ) in 50 m M sodium phosphate at pH 7.2 for 5 min at 37 C. The peroxidase activity was then assessed in PBS containing 100 M 18:2-OOH and 500 M OPD for 15 min at 37 C by measuring the formation of 18:2-OH by HPLC as described under Experimental Procedures. The results are expressed as percentages of activity observed in the absence of modulator, i.e. 10 1 M 18:2-OH. The data are expressed as the means S.D. ( n = 3).

Pf HRP2-FePPIX (1

tered product pattern (Fig. 5B). Also, pretreatment with NaCN, but not azide or aminotriazole, inhibited the peroxidase activity of the PfHRP2-FePPIX complex (Fig. 6), whereas the formation of 18:2=O was unaffected (data not shown). This demonstrates that NaCN selectively inhibited heterolytic cleavage of 18:2OOH, and this rules out the possibility that 18:2-OH is formed via alkoxyl radicals. Exposure to NaCN caused a shift of the Soret band from 411 to 414 nm (Fig. 7) and a broadening of the a- and 13-bands (Fig. 7, inset). The steady-state kinetics of the reaction of PfHRP2-FePPIX with 18:2-OOH in the presence of OPD followed Michaelis-Menten kinetics (Fig. 8), consistent with peroxidase activity of the complex. In general, amines and phenols acted as reducing agents (Table II), suggesting that higher oxidation intermediates of PfHRP2-FePPIX participated in the decomposition of 18:2-OOH (Reactions 3

state intermediate of PfHRP2-FePPIX, we examined whether

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Histidine-rich Protein-2

Plasmodium falciparum

FIG. 7. Spectral changes of Pf HRP2-FePPIX induced by NaCN. PfHRP2 (0.56 AM) was reconstituted with FePPIX (5 AM) in 100 mM sodium phosphate at pH 7.2 for 5 min at 37 C, and the PfHRP2 FePPIX complex then reacted with 10 m M NaCN for 5 min at 37 C. The spectra were taken before ( ) and after (+) addition of NaCN. The inset shows the spectra of PfHRP2 (0.56 AM) reconstituted with FePPIX (50 AM) in the absence and presence of 10 m M NaCN. The results shown are representative of three separate experiments.

FIG. 8. Michaelis-Menten plots of peroxidase activity of PfHRP2-FePPIX. Peroxidase activity of PfHRP2-FePPIX (1 AM PfHRP2 and 1 AM FePPIX) was measured at 37 C for 5 min in 50 m M sodium phosphate (pH 7.2) containing OPD (0200 AM) and 100 AM 18:2-OOH (A) or 100 AM OPD and 18:2-OOH (0200 AM) (B). The production of 18:2-OH was measured by HPLC as described under Experimental Procedures. The data shown represent the average values of an experiment performed in triplicate.

and 4). Notably, ascorbate, a potent reducing agent found in biological tissues including malaria parasite-infected red cells (25), effectively promoted the reduction of 18:2-OOH. Tyrosine failed to act as a reducing agent, and its incubation with PfHRP2-FePPIX in the presence of 18:2-OOH failed to give rise to detectable amounts of di-tyrosine (not shown), indicating that this physiologically readily available amino acid is not likely to participate in redox reactions with PfHRP2-FePPIX. PfHRP2 FePPIX also metabolized 20:4-OOH but not PLPC-OOH (Table III), perhaps because of inaccessibility of the phospholipid hydroperoxide to the histidine residues of PfHRP2. To test the involvement of compound I as a higher oxidation

TABLE II Downloaded from http://www.jbc.org/ by guest on June 23, 2013 Steady-state kinetic parameters of PfHRP2-FePPIX Km and Vmaxvalues for different reductants of PfHRP2-FePPIX were determined by incubating PfHRP2-FePPIX (1 AM PfHRP2, 1 AM FePPIX), 18:2-OOH (100 AM), and the reducing substance indicated (0200 AM) for 5 min at 37 C in 50 m M sodium phosphate buffer, pH 7.2. Accumulation of 18:2-OH was determined by HPLC as described under Experimental Procedures. The data were calculated from respective Michaelis-Menten plots as shown in Fig. 8A and presented as the mean values of an experiment carried out in triplicate.
Reducing substance

V
AM min1

Km
AM

Ascorbate 0.8 Bilirubin conjugate 0.9 FADH 0.3 GSH NAa Guaiacol 3.1 Phenol 1.2 Gallic acid 2.9 Salicylic acid NA Trolox 0.9 Tyrosine NA OPD 1.6 b DPPD 0.8 c ABTS 3.4 NA, not active. DPPD, N,N'-diphenyl-1,4-phenylenediamine. ABTS, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid).
a b

6 33 8 NA 48 366 98 NA 27 NA 9 8 15

TABLE III Steady-state kinetic parameters of PfHRP2-FePPIX Km and Vmaxvalues for different lipid hydroperoxides of PfHRP2 FePPIX were determined by incubating PfHRP2-FePPIX (1 AM HRP2, 1 AM FePPIX), OPD (100 AM), and the lipid hydroperoxide indicated (0200 AM) for 5 min at 37 C in 50 mM sodium phosphate buffer, pH 7.2. Accumulation of lipid hydroxide was determined by HPLC as described under Experimental Procedures. The data were obtained from Michaelis-Menten plots as shown in Fig. 8B and presented as the mean values of an experiment carried out in triplicate.
Hydroperoxide

Vmax
A
M

Km m i n
1

18:2-OOH 20:4-OOH PLPC-OOH NA, not active.


a

1.2 2.9 NAa

6.6 19.0 NA

thioanisole could serve as a reducing agent (26). Consistent with previous studies of peroxidases, the amount of thioanisole sulfoxide formed during the reaction increased with increasing concentration of thioanisole and was comparable with the amount of accumulating 18:2-OH (Fig. 9 A). Also, the accumulation of thioanisole sulfoxide increased with increasing concentrations of 18:2-OOH (Fig. 9 B) and FePPIX (Fig. 9C). Together, these results suggest that the reaction of PfHRP2 FePPIX with 18:2-OOH gives rise to a compound I-like intermediate. Finally, we examined whether complexes of PfHRP2 with other metallo-porphyrins exhibit peroxidase-like activity. Consistent with a previous report (14), PfHRP2-bound metalloporphyrins gave rise to distinct Soret and a- and )9-bands (Fig. 10 and Table IV). In contrast to FePPIX, however, complexes of PfHRP2 with SnPPIX or ZnPPIX, as well as PPIX itself, essentially failed to reduce 18:2-OOH to 18:2-OH or 18:2 =O (Table V).
DISCUSSION

The known function of PfHRP2 is to promote the conversion of FePPIX to hemozoin, the malarial pigment (10). By doing so, PfHRP2 converts the redox-active and toxic FePPIX into insoluble hemozoin crystals that maintain pro-oxidant activity (27). The enhancement of formation of hemozoin by PfHRP2 requires acidic pH (11) and is generally considered to take place in the food vacuole of parasitized red cells where hemoglobin

Plasmodium falciparum Histidine-rich Protein-2 14519


proteolysis occurs (10). However, PfHRP2 is also detected in the cytoplasm and plasma membrane of erythrocytes (12) as well as in the plasma of infected hosts (28). The pH in these compartments is expected to be neutral, i.e. a condition where PfHRP2 binds FePPIX rather than initiates its conversion to hemozoin (11). Thus, the experimental conditions employed here are relevant for sites where the FePPIX crystallizing function of PfHRP2 is limited. The present results suggest that at neutral pH, PfHRP2 modulates the redox activity of FePPIX. Thus, PfHRP2 pro-

F IG . 9. Production of thioanisole sulfoxide by Pf HRP2-FePPIX. A , a mixture containing 3 M Pf HRP2, 3 M FePPIX, and 200 M 18:2OOH was treated with thioanisole at 37 C for 15 min, and the production of thioanisole sulfoxide ( ) and 18:2-OH ( ) was determined by HPLC as described under Experimental Procedures. Similarly, the formation of thioanisole sulfoxide was determined with 3 M FePPIX, 3 M Pf HRP2, 1.6 m M thioanisole, and various concentrations of 18:2- OOH ( B ) and with 02 M FePPIX, 3 M Pf HRP2, 1.6 m M thioanisole, 200 M 18:2-OOH ( C ), respectively. The data are expressed as the means S.D. ( n = 3).

tected ascorbate from degradation induced by FePPIX and transition metals, it inhibited the release of radical intermediates during the metabolism of lipid hydroperoxides mediated by FePPIX, and it increased the peroxidase-like activity of FePPIX. Together, our results indicate a novel function for PfHRP2 as an antioxidant that could be relevant to malaria-infected red cells that are under oxidative stress (3). The binding of FePPIX to PfHRP2 at neutral pH has been characterized previously in detail (13). Up to -50 molecules of FePPIX bind to each PfHRP2 protein and are tightly coordinated to histidine residues. We reasoned that such binding may modulate the redox activity of FePPIX, and our finding that the PfHRP2-FePPIX complex has peroxidase activity supports this view. True peroxidases catalyze the heterolytic reduction of hydroperoxides to the corresponding alcohol (29). In contrast, free FePPIX and FePPIX proteins such as hemoglobin and cytochrome P-450 metabolize hydroperoxides predominantly via homolytic reduction (15). The two pathways can be distinguished by analyzing the stable products formed during the reaction (22). The results of such analysis show that the PfHRP2-FePPIX complex has higher peroxidase activity than FePPIX alone, although the complex is not a true peroxidase in that it also degrades ROOH through homolytic reactions. The enhanced peroxidase activity of the PfHRP2-FePPIX complex relative to free FePPIX is supported by several lines of evidence, in addition to the higher ratio of hetero- to homolytic products (Table I). Thus, the reaction catalyzed by the PfHRP2FePPIX complex followed Michaelis-Menten kinetics, yielded higher turnover numbers for 18:2-OH than did FePPIX, and it was inhibited by CN- that caused an irreversible change in the spectral properties of the protein-bound FePPIX. Also, other metallo-porphyrins were unable to replace FePPIX in the reaction. In addition, the implied suppression of homolytic degradation pathways by PfHRP2 is supported by the co-oxidation experiment; binding of FePPIX to PfHRP2 attenuated the extent of alkoxyl radical formation (Fig. 2). Together, these findings demonstrate the peroxidase-like activity of the PfHRP2- FePPIX complex and strongly suggest that FePPIX is the catalytic center for this activity. Most heme-containing peroxidases contain a neutral amino
TABLE V Peroxidase activity of PfHRP2 reconstituted with different metal-porphyrins Peroxidase activity was determined by incubating the complex of PfHRP2 and the (metal)-porphyrin indicated with 100 M 18:2-OOH and 1 mM OPD in PBS for 15 min at 37 C. 18:2-OH and 18:2=O were then quantified by HPLC as described under Experimental Procedures. The concentrations of 18:2-OH and 18:2=O accumulated were calculated after subtracting the corresponding control value obtained in the absence of OPD. The data are expressed as the means S.D. (n = 3).
(Metal)-porphyrin
A 18:2-OH
M

A 18:2=O
M

F IG . 10. UV-visible spectra of Pf HRP2 reconstituted with various metallo-porphyrins. Pf HRP2 was reconstituted with metal-porphyrin in 200 m M sodium phosphate (pH 7.2) containing 1.1 M Pf HRP2 and a suspension of 100 M FePPIX, SnPPIX, ZnPPIX, or PPIX for 5 min at 37 C. Unbound metallo-porphyrin was then removed by gel filtration through a PD-10 column before the spectra of the reconsti tuted Pf HRP2 (0.56 M ) were recorded in PBS.

FePPIX SnPPIX ZnPPIX PPIX

40.3 1.0 0.6 0.5

3.8 0.7 0.1 0.3

7.3 0.4 0.3 0.2

0.9 0.1 0.1 0.1

Downloaded from http://www.jbc.org/ by guest on June 23, 2013 T ABLE IV Maximal wavelengths of absorption of PfHRP2 reconstituted with different metallo-porphyrins UV-visible spectra were recorded in PBS at room temperature.
Metal-porphyrin
A

Soret

FePPIX

SnPPIX

414 535 565 Ref. 13 392, 404(s)a 539

ZnPPIX

577 This study 401(s), 418 557 586 This study

s, shoulder.

14520 Histidine-rich Protein-2

Plasmodium falciparum

P. Loria, N. Campanale, M. Foley, and L. Tilley, unpublished data.

acid residue on the distal side of the heme that is believed to participate with the distal histidine in the two-electron reduction of the hydroperoxide substrate (30). This view is supported by the loss of peroxidase activity of prostaglandin-endoperoxide synthase-2 in which the relevant neutral amino acid residue glutamine is replaced by valine or arginine (31). Interestingly, PfHRP2 lacks this distal neutral amino acid (8), and this could explain the participation of PfHRP2-FePPIX in homolytic reduction of the hydroperoxide observed in the present study. In this context, it would be interesting to examine the peroxidase activity of PfHRP3, a homologous protein to PfHRP2 that contains asparagine instead of aspartate distal to the heme and close to the distal histidine residue (8). Peroxidases reduce hydroperoxides to the corresponding alcohol with concomitant formation of an intermediate called compound I that has distinct spectral properties (29). Attempts to detect compound I during PfHRP2-FePPIX-mediated reduction of 18:2-OOH failed, even when using rapid scanning spec troscopy (dead time, 0.1 s) at 7 C (data not shown), similar to the situation with methemoglobin and metmyoglobin (32). However, the observed conversion of thioanisole to thioanisole sulfoxide provides indirect evidence for the intermediate formation of compound I (17). In particular, we observed that the production of sulfoxide and 18:2-OH increases in parallel with increasing thioanisole concentration (Fig. 3 A), consistent with the reduction of compound I by thioanisole. The putative involvement of compound I implies that 18:2-OOH can displace a coordinated histidine residue in PfHRP2-FePPIX. We consider the spectral changes observed upon the addition of CN- to PfHRP2-FePPIX (Fig. 7, inset), indicative of a change from hexa- to penta-coordinated heme (29), as precedence for the replacement of a coordinated histidine residue. Ligation of the other histidine to FePPIX appeared to be maintained in the presence of CN-, because the intensity of the Soret band did not change (Fig. 7). That addition of CN - abolished the formation of 18:2-OH but not that of 18:2 =O suggests that CNprevents hydrogen donation from histidine, which is required for peroxidase activity (29). The physiological importance of the peroxidase-like activity of the PfHRP2-FePPIX complex, if any, remains to be established. To be physiologically relevant, PfHRP2-FePPIX and hydroperoxide(s) must co-exist with a suitable reducing substrate, and the peroxidase activity of the complex must effectively compete with other existing peroxidases. The host cytosol of parasitized red cells is a likely site where PfHRP2-FePPIX could act as a peroxidase. Red cells contain ascorbate, a suitable reductant (Table II), and the cellular concentration of the vitamin increases upon malaria infection (33). Also, parasitized red cells contain increased concentrations of polyunsaturated fatty acids and markers of lipid oxidation (34), indicative of an increase in the formation of lipid hydroperoxides. In addition, the activities of red cell glutathione peroxidase and reductase decrease markedly during infection (2), thereby increasing the likelihood of participation of other peroxidases or proteins with peroxidaselike activity. Furthermore, there is evidence that both red cell PfHRP2 (35) and FePPIX co-exist outside the food vacuole. 2 Although PfHRP2-FePPIX has only moderate peroxidase activity when compared with GSH peroxidase, PfHRP2 is concentrated at sites such as underneath the red cell mem-

brane where oxidative damage would be particularly harmful in the parasitized erythrocyte, i.e. if the host cell membrane lyses prematurely, the parasite will not be able to complete the cycle. These considerations together suggest that a role for PfHRP2FePPIX in the metabolism of lipid hydroperoxides in parasitized red cells is feasible and that such a function of PfHRP2 provides a previously unrecognized protection to the parasite against oxidative stress. Additional studies are required to explore this possibility directly.
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35. Papalexis, V., Siomos, M.-A., Campanale, N., Guo, X.-G., Kocak, G., Foley,
M., and Tilley, L. (2001) Mol. Biochem. Pharmacol. 115, 7786

H. (1987) Lipids 22, 5157

AcknowledgmentsWe thank Drs. Andrew Terentis, Stuart Linton and Paul Witting (The Heart Research Institute) and Anthony Kettle (Christchurch School of Medicine, Christchurch, New Zealand) for help ful discussions. The excellent technical assistance of Dr. Aviva Levina (School of Chemistry, University of Sydney) and access to mass spec trometers at the Ray Williams Biomedical Mass Spectrometry Facility (Faculty of Medicine, University of New South Wales) are also acknowledged.
REFERENCES

1. 2.

Francis, S. E., Sullivan, D. J., Jr., and Goldberg, D. E. (1997) Annu. Rev. Microbiol. 51, 97123 Stocker, R., Hunt, N. H., Buffinton, G. D., Weidemann, M. J., Lewis-Hughes, P. H., and Clark, I. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 548551 3. Hunt, N. H., and Stocker, R. (1990) Blood Cells 16, 499526 4. Chou, A. C., and Fitch, C. D. (1981) J. Clin. Invest. 68, 672677 5. Tappel, A. L. (1953) Arch. Biochem. Biophys. 44, 378394 6. Loria, P., Miller, S., Foley, M., and Tilley, L. (1999) Biochem. J. 339, 363 370 7. Goldberg, D. E., Slater, A. F., Cerami, A., and Henderson, G. B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 29312935 8. Wellems, T. E., and Howard, R. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 60656069 9. Panton, L. J., McPhie, P., Maloy, W. L., Wellems, T. E., Taylor, D. W., and Howard, R. J. (1989) Mol. Biochem. Parasitol. 35, 149160 10. Sullivan, D. J., Jr., Gluzman, I. Y., and Goldberg, D. E. (1996) Science 271, 219222 11. Lynn, A., Chandra, S., Malhotra, P., and Chauhan, V. S. (1999) FEBS Lett. 459, 267271 12. Howard, R. J., Uni, S., Aikawa, M., Aley, S. B., Leech, J. H., Lew, A. M., Wellems, T. E., Rener, J., and Taylor, D. W. (1986) J. Cell Biol. 103, 1269 1277 13. Choi, C. Y., Cerda, J. F., Chu, H. A., Babcock, G. T., and Marletta, M. A. (1999) Biochemistry 38, 1691616924 14. Ziegler, J., Chang, R. T., and Wright, D. W. (1999) J. Am. Chem. Soc. 121, 23952400 15. Marnett, L. J., and Kennedy, T. A. (1995) in Cytochrome P450 Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., ed) pp. 4980, Plenum Publishing Corp., New York 16. Ortiz de Montellano, P. R., and Catalano, C. E. (1985) J. Biol. Chem. 260, 92659271 17. Ple, P., and Marnett, L. J. (1989) J. Biol. Chem. 264, 1398313993 18. Mashima, R., Onodera, K., and Yamamoto, Y. (2000) J. Lipid Res. 41, 109 115 19. Mashima, R., Yamamoto, Y., and Yoshimura, S. (1998) J. Lipid Res. 39, 11331140 20. Chan, H. W., and Levett, G. (1977) Lipids 12, 99104 21. Buettner, G. R. (1988) J. Biochem. Biophys. Methods 16, 2740 22. Dix, T. A., and Marnett, L. J. (1985) J. Biol. Chem. 260, 53515357 23. Samokyszyn, V. M., and Marnett, L. J. (1987) J. Biol. Chem. 262, 14119 14133 24. Ortiz de Montellano, P. R., and Grab, L. A. (1987) Biochemistry 26, 5310 5314 25. Stocker, R., Hunt, N. H., Weidemann, M. J., and Clark, I. A. (1986) Biochim. Biophys. Acta 876, 294299 26. Harris, R. Z., Newmyer, S. L., and Ortiz de Montellano, P. R. (1993) J. Biol. Chem. 268, 16371645 27. Green, M. D., Xiao, L., and Lal, A. A. (1996) Mol. Biochem. Parasitol. 83, 183188 28. Parra, M. E., Evans, C. B., and Taylor, D. W. (1991) J. Clin. Microbiol. 29, 16291634 29. Dunford, H. B. (1999) Heme Peroxidases, pp. 1507, John Wiley & Sons, Inc., New York 30. Poulos, T. L., and Kraut, J. (1980) J. Biol. Chem. 255, 81998205 31. Landino, L. M., Crews, B. C., Gierse, J. K., Hauser, S. D., and Marnett, L. J. (1997) J. Biol. Chem. 272, 2156521574 32. Egawa, T., Shimada, H., and Ishimura, Y. (2000) J. Biol. Chem. 275, 34858 34866 33. Stocker, R., Weidemann, M. J., and Hunt, N. H. (1986) Biochim. Biophys. Acta 881, 391397 34. Stocker, R., Cowden, W. B., Tellam, R. L., Weidemann, M. J., and Hunt, N.

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